The invention relates to a method and apparatus for screening large numbers of molecules for biological activities.
Current technology is able to generate large numbers of molecules with potential therapeutic value. These compounds include products of combinatorial or traditional chemistry, natural products, proteins isolated by one- or two-dimensional gel electrophoresis, or compounds secreted from or expressed by natural or genetically modified animal, plant, microbial or fungal cells, or displayed by natural or genetically modified viral or phage particles.
The practice of screening large libraries for a compound having a specific biological activity is routinely used in new drug discovery. Screening methodologies include binding assays or functional assays. A binding assay identifies compounds of interest by affinity to cells or cell products, and may include detection methods such as the use of fluorescent, luminescent, or radioactive labels. Functional assays may also be used, for example, determination of effects on gene expression.
Screening methods have been developed which achieve very high throughputs of test compounds. Such methods are termed Ultra High Throughput Screening (UHTS). The present generation of UHTS machines rely upon essentially serial additions of test compounds, usually one test compound per discrete test well. Test well array densities range from between 96 to 3456 wells per plate. Such UHTS machines require sophisticated technologies to dispense microvolumes of many different fluids to selected locations, and also require that the detecting surface for each test molecule generally be separated from other detecting surfaces within the array.
Visualization of intracellular function using luminescent (fluorescent or bioluminescent) probes has become one of the mainstay techniques in modem cell biology. Using traditional optical microscopes with quantitative detectors in place of the human eye, both the concentration and distribution in the cell of a variety of intracellular molecules of interest can be measured. While luminescent probes can be measured in large populations of cells using other techniques, imaging is critical for studying single cells or small populations of cells. Most of these probes can be introduced non-invasively into cells and will, depending on the detection system, allow characterization of cellular events in high temporal resolution (microseconds to seconds) and high spatial resolution (nanometers to micrometers). This probe technology, in combination with the technology of cellular imaging, has enabled monitoring of complex, cross-reacting intracellular events not previously accessible by conventional techniques. The use of luminescent probes for cell-based screening is described in PCT publication WO 97/45730.
There is a need to develop a method which allows simultaneous screening of large numbers of test compounds for biological activity and potential therapeutic use while avoiding the complications associated with dispensing multiple microvolumes of many different fluids.
The invention is directed to a screening method which eliminates the need for delivering microfluid volumes and allows simultaneous parallel screening of large numbers of test compounds. Accordingly, the invention is drawn to a method for screening test compounds for bioactivity, by contacting an array of test compounds with a detector layer capable of detecting bioactivity, wherein a cell response is indicative of bioactivity.
In the method of the invention, a detector layer may be comprised of physiologically viable cells, which in a specific embodiment form a monolayer. The detection step may comprise a change in a fluorescence or luminescence property of the cell. In a specific embodiment, detection is determined with an illumination system capable of exciting the fluorescence of the detector layer with any of a number of previously selected wavelengths with defined order and of defined time duration.
In related embodiments, the detector layer may be scintillant plastic, a pH sensing surface, or a temperature sensitive surface. One example of a scintillant plastic is Cytostar plates (Amersham). Cells are grown on top of the scintillant plastic, radio-labelled ligand is applied to the cells, and then displaced by test compounds which are competitive to those ligand receptors. The signal is a reduction in scintillation at the point in the detector layer corresponding to an xe2x80x9cactivexe2x80x9d ligand in the array of test compounds. A pH sensing surface may also be used (Molecular Devices Corp.) incorporated into the solid support upon which cells in the detector layer are growing. Many changes in cellular activity result in changes in proton extrusion from cells, and hence localised changes in the pH of medium around responding cells. Temperature sensing surface, such as a sensitive calorimetric or fluorescent liquid crystal layer, may also be incorporated into the substrate upon which the cells are growing. Such a detector layer is useful for metabolic responses which give rise to changes in heat flux from cells.
In specific embodiments, the detector layer is supported by an optically clear substrate. Further, the detector layer may be held stationary in the field of view of the lensing sytem and camera and the sample surface is moved into contact with the detector layer during the course of measurement. In another embodiment, the sample surface is held stationary in the field of view of the lensing system and camera and the detector layer is moved into contact with the sample surface during the course of measurement.
The test compounds screened in the method of the invention may be generated by a variety of methods known to the art, including those generated on a solid support by combinatorial chemistry, or by one- or two-dimensional gel electrophoresis.
The method of the invention is a high throughput system for parallel screening of a large number of test compounds. In one embodiment of the method of the invention, 96 to 10,000 test compounds are simultaneously screened for bioactivity in an assay; in a more specific embodiment, 96 to 6,144 test compounds are simultaneously screened for bioactivity.
In a related embodiment, the invention is drawn to a method for screening test compounds for bioactivity, by (1) contacting a solid support comprising an array of test compounds with a liquid layer, wherein the liquid layer is in immediate contact with a detector layer and wherein each test compound comes into contact with a localized portion of the liquid layer; and (2) registering a response of the detector layer to the test compound, wherein a bioactive test compound is identified.
In a related embodiment, the invention is drawn to a method for high throughput screening of test compounds for bioactivity, comprising (a) contacting a solid support comprising an array of multiple test compounds with a detector layer, wherein each test compound comes into contact with a localized liquid which is in contact with the detector layer; and (b) detecting a response of the detector layer to the test compound, wherein a response is indicative of a bioactive compound.
In another related embodiment, the invention is drawn to a method for simultaneously exposing an array of test compounds with a detector layer, comprising the steps of (a) contacting an array of test compounds on a solid substrate with a porous membrane which is in contact with a liquid layer surrounding a detector layer, (b) allowing the test compounds to move via the porous membrane to the liquid layer surrounding the detector layer, and (c) detecting a response in the detector layer where the detector layer comprises a layer of physiologically viable cells.
In another related embodiment, the invention is drawn to a method for simultaneously exposing an array of test compounds with a detector layer, comprising the steps of (a) contacting an array of test compounds held on a porous membrane or non-porous substrate with a liquid layer overlaying a detector layer, (b) allowing the test compounds to move from the porous membrane or non-porous substrate into the liquid layer overlaying the detector layer, and (c) detecting a response in the detector layer where the detector layer comprises a layer of physiologically viable cells.
The invention further features an apparatus for screening an array of test compounds for bioactivity, comprising (a) a solid support comprising an array of test compounds, (b) a porous membrane, and (c) a detector layer, wherein a liquid layer is between the porous membrane and detector layer, and wherein the test compounds contact the detector layer by movement via the porous membrane.
One advantage of the method of the invention is that it allows massively parallel screening of a large array of test compounds for biological activity. When physiologically viable cells are the detector layer of the invention, they are maintained under physiologically viable conditions. Provision of these conditions requires the use of solutions able to supply essential nutrients and buffer pH changes normal to the continued growth of physiologically viable cells. Such solutions may be complete cell culture media (i.e. any of those commercially available, for instance from Life Technologies Ltd.), optionally supplemented with antibiotics and serum preparations for optimal cell growth conditions. Buffer solutions may also be of the type known as xe2x80x9cchemically definedxe2x80x9d. Cells will also require controlled temperature conditions, in the range 20xc2x0 to 37xc2x0 C., and the provision of gases essential to continued cell growth and maintenance of buffer capacity (O2, and optionally 5% CO2, depending on the type of buffer being used).
These and other objectives, advantages, and features of the invention will become apparent to those persons skilled in the art upon reading the details of the method as more fully described below.